Dissecting the relative contribution of ECA3 and group 8/9 cation diffusion facilitators to manganese homeostasis in Arabidopsis thaliana

Abstract Manganese (Mn) is an essential micronutrient for plant growth but becomes toxic when present in excess. A number of Arabidopsis proteins are involved in Mn transport including ECA3, MTPs, and NRAMPs; however, their relative contributions to Mn homeostasis remain to be demonstrated. A major focus here was to clarify the importance of ECA3 in responding to Mn deficiency and toxicity using a range of mutants. We show that ECA3 localizes to the trans‐Golgi and plays a major role in response to Mn deficiency with severe effects seen in eca3 nramp1 nramp2 under low Mn supply. ECA3 plays a minor role in Mn‐toxicity tolerance, but only when the cis‐Golgi‐localized MTP11 is non‐functional. We also use mutants and overexpressors to determine the relative contributions of MTP members to Mn homeostasis. The trans‐Golgi‐localized MTP10 plays a role in Mn‐toxicity tolerance, but this is only revealed in mutants when MTP8 and MTP11 are non‐functional and when overexpressed in mtp11 mutants. MTP8 and MTP10 confer greater Mn‐toxicity resistance to the pmr1 yeast mutant than MTP11, and an important role for the first aspartate in the fifth transmembrane domain DxxxD motif is demonstrated. Overall, new insight into the relative influence of key transporters in Mn homeostasis is provided.

Mn deficiency is one of the most frequently occurring nutritional disorders in cereal crops and is more common on sandy and calcareous soils (Jiang, 2006). Symptoms often include stunted growth, interveinal chlorosis, and slack and soft leaves, due to a reduced content of fructans and structural carbohydrates (Pearson & Rengel, 1997).
Increased transpiration and decreased water-use efficiency, associated with a decrease in epicuticular wax, have also been reported under Mn deficiency (Hebbern et al., 2009). Toxic levels of Mn can be equally detrimental to plant development, with Mn bioavailability increasing in acidic soils (Adams, 1981). Mn can replace magnesium (Mg) from key active sites to inhibit enzymatic reactions under these conditions (Bock et al., 1999). Secondary iron (Fe) deficiency can also be induced under Mn toxicity, particularly on calcareous soils where Fe availability is also limiting (Eroglu et al., 2016;Marschner, 2012).
Mn toxicity symptoms include browning and cracking of roots, chlorosis of the leaf, and brown spots on mature leaves caused by accumulations of oxidized Mn and phenols, leading to losses in agricultural yield (Fecht-Christoffers et al., 2003;Williams & Pittman, 2010).
It is therefore important to develop new and innovative approaches to target the agricultural yield losses associated with both Mn deficiency and toxicity (Williams & Pittman, 2010). Although fertilizers can be applied to help overcome Mn deficiency, their efficacy can be reduced by oxidation, and more sustainable approaches are required. There is great interest in breeding more nutrient-efficient crops that could improve productivity on nutrient-poor soils. Transgenic technologies also provide a wealth of opportunities to modify micronutrient efficiency and nutritional composition. Understanding the homeostatic mechanisms that control and balance levels of essential nutrients across the plant is an important starting point (Salt & Williams, 2009). Membrane transporters play a vital role in controlling metal uptake and distribution around the plant while regulating levels to avoid toxicity.
Recent years have seen great progress in the molecular characterization of transition metal transporters in Arabidopsis. The natural resistance-associated macrophage protein, NRAMP1, provides highaffinity Mn uptake at the plasma membrane (Cailliatte et al., 2010), and the Fe-deficiency-regulated IRT1 also has affinity for Mn uptake (Korshunova et al., 1999;Vert et al., 2002). To prevent cytoplasmic toxicity, Mn is sequestered mainly in the vacuole but has also been found in the chloroplasts, mitochondria, Golgi, and endoplasmic reticulum (ER) (Williams & Pittman, 2010). Tonoplast-localized transporters such as Ca 2+ and Mn 2+ /H + antiporters, CAX2 and CAX5, are involved in vacuolar influx of Mn (Connorton et al., 2012;Hirschi et al., 2000) as is MTP8, an MTP (metal tolerance protein), which is also proposed to be important under Fe deficiency (Eroglu et al., 2016). MTP8 is also responsible for Mn and Fe accumulation in seeds (Chu et al., 2017;Eroglu et al., 2017). In contrast, NRAMP3 and NRAMP4 serve to redistribute Mn from the vacuole to the chloroplasts for its role in photosynthesis (Lanquar et al., 2005(Lanquar et al., , 2010, whereas the trans-Golgi network-localized NRAMP2 is important under Mn deficiency (Alejandro et al., 2017;Gao et al., 2018). Recently, PAM71 (encoded by photosynthesis affected mutant 71) and CMT1 (encoded by chloroplast manganese transporter 1) have been identified as thylakoid membrane and inner envelope membrane proteins, respectively, for Mn uptake in the chloroplast (Eisenhut et al., 2018;Schneider et al., 2016;Zhang et al., 2018). PAM71 and CMT1 belong to a small five-member protein family in Arabidopsis, the other members of which, PML3 (photosynthesis-affected mutant 71 like 3) and PML4/5, are involved in Mn transport at the Golgi and endoplasmic reticulum, respectively (Hoecker et al., 2020;Yang et al., 2021).
Aside from a few examples such as NRAMP (Gao et al., 2018;Lanquar et al., 2010) and CAX transporters (Connorton et al., 2012), most of the players involved in Mn homeostasis in plants have been studied independently and often under different conditions. It is important to directly compare their relative contribution to Mn homeostasis to inform strategies for sustainable crop improvement.
This study focuses on Arabidopsis ECA3, a P 2A -type ATPase (Aslam et al., 2017;Barabasz et al., 2011;Li et al., 2008;Mills et al., 2008;Pittman et al., 1999) and Group 8/9 MTP members, MTP8, MTP10, and MTP11 (Delhaize et al., 2007;Eroglu et al., 2016;Peiter et al., 2007;Ricachenevsky et al., 2013) as well as NRAMP1 and NRAMP2 (Alejandro et al., 2017;Cailliatte et al., 2010;Gao et al., 2018). Although it is clear that MTP11 is essential for alleviating Mn toxicity (Delhaize et al., 2007;Peiter et al., 2007), studies on ECA3 have indicated roles in Mn deficiency (Mills et al., 2008) but also in alleviating Mn toxicity (Li et al., 2008). To resolve this, we characterized novel eca3 mtp11 double mutants under different Mn extremes to clarify the importance of ECA3 and MTP11 in these processes. We also address the previously disputed subcellular localization of ECA3 and MTP11 (Delhaize et al., 2007;Li et al., 2008;Mills et al., 2008;Peiter et al., 2007). Further, we assessed the contribution of the Mn-MTPs in Mn homeostasis, generating and directly comparing novel double and triple mutants for mtp8, mtp10, and mtp11. From this and localization studies, a role for MTP10 at the Golgi in alleviating Mn toxicity was identified. Additionally, although NRAMP1 and NRAMP2 are known to play a role in Mn deficiency (Cailliatte et al., 2010;Gao et al., 2018), here the relative importance of each NRAMP and ECA3 was investigated by comparing single, double, and triple nramp1, nramp2, and eca3 mutants under Mn-deficiency conditions. This study provides further understanding of key transporters and their relative importance in contributing to Mn homeostasis under low and high Mn.

| RESULTS
A specific aim of this study was to clarify the importance of ECA3 under Mn deficiency and toxicity, as it is currently unclear where it has its major contribution. The first part of the study investigates its role under Mn deficiency. NRAMP1 and NRAMP2 transporters have important roles in Mn deficiency, and so a comparison of mutants was undertaken to investigate the relative effects when ECA3, NRAMP1, and NRAMP2 were knocked out.
2.2 | Mn-sensitivity of eca3 mtp11 double mutants under two Ca regimes MTP11 has a major role in mitigating Mn toxicity (Delhaize et al., 2007;Peiter et al., 2007), but the role of ECA3 is less certain. Therefore, it is important to compare their relative contributions more directly. Double mutants were generated and compared to single mutants to investigate their response under different Mn conditions. T-DNA insertion mutants eca3-1 and eca3-2 (Mills et al., 2008) were both crossed with mtp11-1 (Delhaize et al., 2007), and homozygous double mutants, eca3-1 mtp11-1 and eca3-2 mtp11-1, were isolated.
RT-PCR was used to confirm double mutants at the RNA level ( Figure S2A).
Mutants were compared across a range of Mn conditions from deficiency (0 Mn supplied) to excess Mn to clarify the role of ECA3 and MTP11 in Mn homeostasis ( Figure 2). Because ECA3 is proposed to transport Ca as well as Mn, and eca3 mutants were previously shown to be extremely stunted on low-Ca and low-Mn medium (Mills et al., 2008), we employed two calcium (Ca) regimes. Mutants were grown on half-strength MS medium (1/2 MS) at standard Ca (1.495mM) and low Ca (100 μM). All mutants grew similarly to wild-type (WT) plants under basal Mn conditions (50 μM Mn). Under Mn-deficiency conditions (0 μM Mn) at both Ca levels, whereas eca3-1 and eca3-2 single mutants displayed hypersensitivity, mtp11-1 performed similarly to WT. Under these conditions, the eca3 mtp11 double mutants performed similarly or even better than the eca3 single mutants (Figure 2a,b). A further mutant, eca3-4, was also sensitive to Mn deficiency (stunting and chlorosis), supporting findings by Mills et al. (2008) that ECA3 has a crucial role in Mn nutrition. Under elevated Mn, mtp11-1 displayed a hypersensitive phenotype (stunting and chlorosis), which was apparent at lower Mn concentrations under the low Ca regime (100 μM Mn compared with 300 μM Mn).
Importantly, the eca3-2 mtp11-1 and eca3-1 mtp11-1 double mutants displayed a more severe sensitivity to Mn toxicity than mtp11-1 under both standard and low Ca (Figure 2a,b). At elevated Mn, eca3-1 and eca3-2 single mutants generally responded similarly to WT. In a separate experiment, eca3-4 also showed no sensitivity to Mn toxicity under either Ca regime ( Figure S3), contradicting reports by Li et al. (2008) that showed eca3-4 displaying inhibition at just 50 μM Mn. Therefore, a role in alleviating toxicity for ECA3 is only apparent when MTP11 is non-functional.
It was noticeable that the Mn-toxicity-dependent phenotype of mtp11 and eca3 mtp11 mutants is observed at lower Mn concentrations under the low Ca regime, than under the basal Ca regime (Figure 2a,b). To further investigate this Mn/Ca antagonism, Mn was provided as 100 μM, with Ca ranging from 100 to 1495 μM ( Figure 2c). Our results show that 100 μM Mn becomes increasingly inhibitory to sensitive genotypes (mtp11-1 and eca3-2 mtp11-1) when Ca is lowered, with significant stunting compared with WT below 300 μM Ca. At this lower range, the eca3-2 mtp11-1 double mutant is significantly more inhibited than the mtp11-1 single mutant.

| ECA3 and MTP11 target different Golgi compartments
Although the role of MTP11 in contributing to Mn detoxification is clear, its localization is uncertain and has been reported to target either the trans-Golgi network (Peiter et al., 2007) or the pre-vacuolar compartment (PVC; Delhaize et al., 2007). Similarly, ECA3 has been proposed to target the Golgi (Mills et al., 2008) or the PVC or another endosomal compartment (Li et al., 2008). Here when stably expressed in Arabidopsis, we show that MTP11 displays a punctate expression pattern (Figure 3a  I G U R E 1 Comparison of nramp1-1, nramp2-5, eca3-1 single, double, and triple mutants under Mn deficiency. Comparison of Columbia 8 wild type (WT) with single mutants of eca3-1, nramp1-1, and nramp2-5 and corresponding double mutants (a) eca3-1 nramp1-1, (b) nramp1-1 nramp2-5, (c) eca3-1 nramp2-1, and (d) nramp1-1 nramp2-5 eca3-1 when grown for 21 days on ½ MS supplemented with either 0 or 50 μM MnSO 4 . Data show mean fresh weight (FW) per seedling (±SE) calculated for six plates per condition, with four seedlings per genotype per plate. Statistical significance was assessed with two-way ANOVA and Tukey's post hoc test. Means not sharing a letter at each individual concentration are significantly different. Photographs display representative plant growth at basal (50 μM) and deficient (0 μM) Mn concentrations. White bar = 1 cm. compared directly, ECA3 and MTP11 show areas of distinct but incomplete overlap (Figure 3m-p); correspondingly, ECA3 displays strong overlap with trans-Golgi marker ST::RFP (Figure 3q-t). It appears, therefore, that ECA3 targets the trans-Golgi, whereas MTP11 targets the cis-Golgi.

| Determining the contribution of Group 8/9 MTPs to Mn homeostasis
The second key aim of this study was to determine the relative contribution of the Arabidopsis Mn-MTPs in Mn homeostasis. An updated phylogenetic tree for putative plant Mn-MTPs is presented in Figure S4. This analysis includes proteins that have not been reported previously: members from Brassica rapa, identified from Phyto-zome9.1, and the putative Hordeum vulgare (barley) HvMTP11, identified by searching the International Barley Sequencing Consortium (Table S2). Phylogenetic analysis confirms the clustering of MTP8 proteins in Group 8, separately from those in Group 9 (MTP9, MTP10, and MTP11). It appears that most monocots, including rice, barley, and sorghum, possess two MTP8 Group 8 members, whereas Arabidopsis possesses only one. Arabidopsis contains three Group 9 MTPs: MTP11 and more divergent MTP9 and MTP10. Monocots, meanwhile, contain two MTP11 sequences but only one other MTP9. All proteins clustering into Group 8 and Group 9 are confirmed to carry the MTP signature sequence and two DxxxD domains on putative transmembrane domains (TMDs) two and five, characteristic of the Mn-MTPs . Arabidopsis MTP1, MTP6, and MTP7 are included in the phylogenetic analysis, clearly clustering separately into Groups 1, 6, and 7, respectively (Gustin et al., 2011).
Sequences for MTP8 and MTP10 are identical to information listed on The Arabidopsis Information Resource (TAIR), whereas MTP11 corresponds to that previously published (Delhaize et al., 2007;Peiter et al., 2007). Two gene models are listed on TAIR for the MTP9 coding sequence, differing in 36 bases. The cDNA cloned in this study corresponded to the MTP9.1 model, here referred to simply as MTP9. The corresponding intron/exon structures are shown in Figure S5

| Generation and analysis of Group MTP8/9 mutants
To determine the contribution to Mn homeostasis, we isolated single T-DNA insertion mutants for MTP8 (mtp8-1 and mtp8-2 in the Columbia background; described in Eroglu et al., 2016) and MTP10 (mtp10-1 and mtp10-2, in the Columbia background). Confirmed insertion sites are highlighted on the intron/exon diagram structures in Figure S5. We also generated a series of double and triple mutants for mtp8, mtp10, and mtp11; confirmation of these mutants is shown in Figure S2B-E. At the time, there was no mtp9 mutant available in the same background.
When tested under Mn deficiency, none of the single mutants were significantly affected compared with WT ( Figure S7). A direct comparison for mtp8-2, mtp11-1, and the corresponding double mutant is shown, across a range of Mn treatments, under both basal Ca ( Figure 4) and low Ca ( Figure S7A,B). Both single mutants were inhibited by Mn toxicity; mtp11-1 was more susceptible than mtp8-2, becoming hypersensitive at lower concentrations and displaying greater levels of stunting and chlorosis. Sensitivity to elevated Mn was exacerbated by low Ca conditions. The mtp8-2 mtp11-1 double mutant displayed greater sensitivity than either of the singles under both Ca regimes. Also, under low Ca conditions, the germination of mtp8-2 mtp11-1 dropped from 100% at 50 μM Mn to 4% at 150 μM Mn, whereas the germination of the other genotypes remained unaffected ( Figure S8).
The role of MTP10 in Mn homeostasis has not previously been reported. Knocking out MTP10 in addition to mtp11, in the mtp10 mtp11 double mutants, had no additional effect compared to growth of the single mtp11 mutant ( Figure S7C). Although the average FW for the mtp10 mtp11 mutant was consistently lower than mtp11 under Mn toxicity, this was not significant. Similarly, knocking out MTP10 in addition to mtp8, in the mtp8 mtp10 double mutant, had no additional effect compared the single mtp8 mutant ( Figure S7D).
However, an underlying contribution of MTP10 to alleviating Mn toxicity did become apparent in the triple mutant, mtp8-2 mtp10-1 mtp11-1, which was significantly more inhibited by Mn toxicity than the double mtp8-2 mtp11-1, under both Ca regimes (Figures 4b and S7B). The triple mutant was inhibited at 200 μM Mn F I G U R E 2 eca3 mtp11 double mutants show increased susceptibility to Mn toxicity when grown under both Ca regimes. (a,b) Comparison of Col8 WT, mtp11-1 and either (a) eca3-2 and eca3-2 mtp11-1 or (b) eca3-1 and eca3-1 mtp11-1 under two Ca regimes. Plants were grown for 19 days on ½ MS supplemented with a range of MnSO 4 concentrations, and either (a) 1495 μM Ca (basal Ca) or (b) 100 μM Ca (low Ca). (c) Increasing Ca alleviates Mn toxicity. Plants were grown on ½ MS containing 100 μM MnSO 4 with a range of CaCl 2 concentrations for 20 days. Data show mean FW (mg) per seedling (±SE) calculated for six plates, with four seedlings per genotype per plate. Statistical significance was assessed with two-way ANOVA and Tukey's post hoc test. (a,b) Means not sharing a letter at a particular condition are significantly different. (c) *, significantly smaller than WT; #, significantly smaller than mtp11-1. Photographs display representative growth across different conditions. White scale bar = 1 cm. See also Figures S2 and S3 and Table S1.
F I G U R E 4 Increased susceptibility to Mn toxicity of mtp8 mtp11 double mutant and mtp8 mtp10 mtp11 triple mutant under basal Ca conditions. Average fresh weight (FW) per seedling of Col8 WT with either (a) mtp11-1, mtp8-2, and mtp8-2 mtp11-1 or (b) mtp11-1, mtp8-2 mtp11-1, and mtp8-2 mtp10-1 mtp11-1. Plants were grown on ½ MS supplemented with a range of MnSO 4 concentrations for 21 days. Data show mean FW (mg) per seedling (±SE) calculated for six plates, with four seedlings per genotype per plate. Statistical significance was assessed with two-way ANOVA and Tukey's post hoc test. Means not sharing a letter at each individual concentration are significant. Photographs display representative plant growth across different Mn concentrations. White bar = 1 cm. See also Figures S4-S7. Mn-induced inhibition of the Fe-deficiency response machinery (Eroglu et al., 2016). A stunted, chlorotic phenotype was seen for mtp8-1 and mtp8-2 under low Fe availability induced by high pH (½ MS plates with 28 μM Fe pH 6.7; Eroglu et al., 2016). In order to determine whether other MTP members are involved in this proposed mechanism, mtp8-2, mtp11-1, single and double mutants were grown on plates under the same conditions (Figure 6a), whereas further mtp single, double, and triple mutants were grown on limed soil where pH was raised to 7.2 ( Figure 6b). In both experiments, plants with MTP8 mutations were stunted and chlorotic. The other single mutants were unaffected compared with WT, and there was not a marked difference in double and triple mutants with a non-functional MTP8 to the mtp8-2 single mutant ( Figure 6), indicating that only MTP8 plays the major role under these conditions. 2.6 | MTP8 targets the tonoplast in planta whereas MTP10 is Golgi-localized Previously, MTP8 has been localized to the tonoplast when expressed in mesophyll protoplasts (Eroglu et al., 2016) and Nicotiana benthamiana leaves (Zhang et al., 2021). Here, we take a step forward and which were formerly only tested up to 6 and 3 mM Mn, respectively (Eroglu et al., 2016;Peiter et al., 2007), and demonstrates MTP10 is F I G U R E 6 MTP8 is the primary Mn-MTP involved in alleviating symptoms under low Fe/high pH conditions. (a) Average fresh weight (FW; mg) per seedling and average chlorophyll (chl; μg) per mg FW of Col8 WT, mtp11-1, mtp8-2, and mtp8-2 mtp11-1. Plants were grown for 23 days on ½ MS (modified Eroglu regime) supplemented with either 28 μM or 100 μM FeNaEDTA (Fe28 and Fe100, respectively), buffered to pH 5.5 or 6.7 with NaOH. Data  equally effective in rescuing pmr1. Tagging MTP10 C-terminally with GFP interferes with its ability to rescue pmr1 above 5 mM Mn ( Figure 9a). In order to determine MTP8/10/11 specificity for Mn transport, each MTP was also expressed in Zn-, cobalt-(Co) and Fe-sensitive yeast mutants. None of the MTPs appear able to effectively restore tolerance to Zn-and Co-sensitive zrc1cot1 (Figure 9c), whereas only non-tagged MTP11 is able to partially restore tolerance of ccc1 to Fe (Figure 9b). Previously, MTP8 has been shown to rescue ccc1 (Chu et al., 2017), which is in contrast to what we observed here.
To explore the importance of the conserved DxxxD domain of TMD 5 in MTP function, site-directed mutants were generated to substitute the DxxxD of MTP8 and MTP11 for HxxxD, as found in Zn-transporting MTP1 (MTP8-D258H and MTP11-D249H). Expression in yeast indicates that this substitution abolishes Mn transport in both MTP8 and MTP11 compared with the non-mutated form.
D249H in MTP11 appears to confer a very slight resistance to Zn compared with non-mutated MTP11 in zrc1cot1; there was no effect of these mutations on Co or Fe transport (Figure 9a-c). A hypothetical tertiary structure of MTP8 and MTP11 was generated using EcYiiP, the only CDF crystallized to date, as a homology model template ( Figure 9d). The DxxxD/HxxxD domains of EcYiiP TMDs 2 and 5, respectively, are reported to form a Zn-binding site coordinated by the three aspartate and single histidine ions (Lu & Fu, 2007;Lu et al., 2009). Based on this model, the DxxxD domains of MTP8 and MTP11 are also predicted to form a potential ion-binding pore within the F I G U R E 7 MTP8 and MTP10 target the tonoplast and trans-Golgi in planta, respectively. (a-d) MTP8::GFP targets the tonoplast when stably expressed in 7-day-old Arabidopsis seedlings. (a) MTP8::GFP in root cells (green signal; filled arrow highlights double vacuole) with (b) cell walls stained with propidium iodide (red signal; unfilled arrow); (c) merged image of (a) and (b). (d) Merged 3D Z-stack of guard cell and neighboring epidermal cells, with MTP8::GFP (green; unfilled arrow) and chloroplast autofluorescence (red signal; filled arrow). MTP8 signal does not enclose the chloroplast, which is characteristic of tonoplast localization. (e) MTP8::GFP shows tonoplast localization when transiently expressed in tobacco epidermal cells. Formation of two transvacuolar strands is indicated (filled and unfilled arrows). (f-k) MTP10::GFP targets the trans-Golgi. Stable expression of MTP10::GFP in 7-day-old Arabidopsis seedlings, displaying punctate fluorescent expression in (f) epidermal leaf cells and (g,h) root cells. Punctate signal is clearly distinct from developing vacuoles, marked with small white arrow. (i) Transient expression of MTP10::GFP in tobacco epidermal cells (green signal), colocalizing with trans-Golgi marker sialyl transferase (ST::RFP; red signal). (k) Merged image of (i) and (j); filled arrows indicate overlapping signal. White bar = 10 μm. See also Movie S1.

| The major function of ECA3 is in Mn deficiency
A major aim of this investigation was to further understand the ways in which ECA3 and MTP11 transporters contribute to Mn transport and homeostasis in Arabidopsis. MTP11 has been shown to be involved in toxicity tolerance (Delhaize et al., 2007;Peiter et al., 2007), but the role of ECA3 is more controversial, with claims that it has a role in either Mn deficiency (Mills et al., 2008) or toxicity (Li et al., 2008). This was addressed here by directly comparing the growth of single mutants at different levels of Mn supply and also generating a double mutant in which both genes were non-functional.
The eca3-1, eca3-2, and eca3-4 mutants displayed clear growth defects under Mn deficiency, both under low and standard Ca, thus confirming an important role for ECA3 during low Mn conditions (Mills et al., 2008). Knocking out mtp11 as well under these conditions did not add to this detrimental effect; if anything, a slight improvement was observed, which could indicate MTP11 may be sequestering the trace amount of Mn that is available for important processes elsewhere. Thus, ECA3 plays an important role in contributing positively to Mn efficiency. A schematic for the roles of ECA3 and MTP11, as well as the other transporters investigated in this study, is shown in Figure 10. Previous models have suggested that under Mn-deficiency conditions, NRAMP1 is responsible for Mn import at the plasma membrane and NRAMP2 exports Mn from the TGN, where it is then used in other subcellular compartments, including the vacuole and chloroplasts via NRAMP3 and NRAMP4 (Alejandro et al., 2017). NRAMP2, NRAMP3, and NRAMP4 act together in the process to provide Mn to photosystem II (PSII) (Alejandro et al., 2017). We show that ECA3 is important under Mn deficiency and propose it sequesters Mn into the trans-Golgi for incorporation into and proper glycosylation of important enzymes and proteins (shown in green). Under Mn-toxic conditions, we use our protein localization data to hypothesis a further model. MTP8 is involved in sequestering toxic Mn from the cytoplasm into the vacuole. Meanwhile, MTP10 and MTP11, which sequester Mn into the cis-and trans-Golgi, respectively, initiate vesicular trafficking to the plasma membrane for efflux from the cell, as proposed by Peiter et al. (2007). ECA3 also has a minor role (only seen when MTP11 is non-functional) in alleviating Mn toxicity, where it may sequester toxic Mn from the cytoplasm. Red, involved in sequestration to alleviate Mn toxicity; yellow, involved in sequestration or uptake to alleviate Mn deficiency. ECA3 is proposed to be involved predominantly in Mn deficiency with a minor role under toxicity. Arrow shows proposed direction of Mn transport with respect to the membrane. deficiency. Additionally, the corresponding double mutants are each additive under Mn deficiency, indicating distinct functions of these transporters. Moreover, the triple nramp1-1 nramp2-5 eca3-1 triple mutant showed further additive sensitivity in comparison to the nramp1-1 nramp2-5 double mutant. The additive sensitivities correspond with the findings that each protein targets a different subcellular membrane, with NRAMP1 targeting the plasma membrane (Cailliatte et al., 2010), NRAMP2 targeting the TGN (Alejandro et al., 2017;Gao et al., 2018), and ECA3 targeting the trans-Golgi ( Figure 10).
3.2 | ECA3 has a minor role in alleviating Mn toxicity, but only when MTP11 is non-functional Li et al. (2008) claimed a role for ECA3 in Mn detoxification, reporting strong inhibition of root growth in eca3-4 mutants (50%-60% inhibition compared with WT at 50 μM Mn). We compared all three eca3 mutants together with WT but did not observe any significant inhibition compared with WT under 50 μM Mn, nor at higher concentrations. mtp11-1 is highly sensitive to elevated Mn, and so it is clear that MTP11 is more important than ECA3 under Mn toxicity.
However, greater levels of stunting and chlorosis are observed in the eca3 mtp11 double mutants compared with mtp11-1, suggesting ECA3 does play a minor role in alleviating Mn toxicity, which is only apparent when MTP11 is non-functional. These findings may suggest ECA3 and MTP11 participate in Mn homeostasis at different pathways. Here, we transiently co-expressed MTP11 and ECA3 in tobacco, together and with organelle markers, demonstrating that ECA3 targets the trans-Golgi, whereas MTP11 targets the cis-Golgi ( Figure 10). This finding favors reports by Peiter et al. (2007), who hypothesized that MTP11 aids Golgi-based Mn accumulation, leading to vesicular trafficking and exocytosis as a route for Mn detoxification. The Golgi localization of ECA3 also favors findings by Mills et al. (2008). This could imply that the primary role of ECA3 in Mn homeostasis is to alleviate deficiency by supplying Mn to key enzymes in the Golgi. However, this Golgi compartmentalization may also be beneficial under elevated Mn, sequestering potentially toxic Mn from the cytoplasm. This minor role is only apparent when MTP11 is non-functional, perhaps due to different thresholds of the trans-and cis-Golgi compartments for Mn accumulation. This study also highlights the importance of the Golgi in both extremes of Mn nutrition in plants.

| Susceptibility to Mn depends on external Ca concentration
Here, we have shown that the level of Ca supplied also has an effect on the concentration at which Mn toxicity symptoms are observed.
When exposed to 100 μM Mn in combination with standard Ca levels (1.495 mM Ca), growth was not detrimentally affected; reducing the Ca concentration caused this Mn concentration to become increasingly toxic to all genotypes but particularly to mtp11-1 and eca3 mtp11. The greater sensitivity of the eca3 mtp11 mutant compared with mtp11-1 was clearly seen. Similarly, we also observed a Mndependent reduction in germination in mtp8 mtp11 and mtp8 mtp10 mtp11 that was apparent under the low Ca conditions. Addition of Ca can reduce Mn uptake and toxicity in young tomato plants (Gunes et al., 1998), peanut plants (Bekker et al., 1994), and barley (Alam et al., 2006). Elevated Ca can also lead to greater Fe accumulation in barley by alleviating Mn-induced Fe deficiency (Alam et al., 2006) (Ghanotakis et al., 1985). Ca can be replaced with other metals in this protein and retain the core structure, but Ca is essential for proper function due to its role in organizing the water network surrounding the protein (Lohmiller et al., 2012). Importantly, we show that MTP8 is the only Mn-MTP involved in providing this tolerance; the other single mtp mutants remain unaffected and knocking out MTP10 and MTP11 in addition to MTP8 has no additional marked effect.

| Overexpression of MTP8 in Arabidopsis conferred enhanced tolerance to Mn toxicity
When we stably expressed MTP8-GFP in Arabidopsis, and transiently in tobacco cells, fluorescence is characteristic of the tonoplast. The MTP8 signal runs continuously, internally to chloroplast autofluorescence, and forms transvacuolar strands to enable passage of the Golgi and cytoplasmic contents across the cell. A tonoplast localization has previously only been reported when MTP8 is transiently expressed in mesophyll protoplasts and tobacco leaves (Eroglu et al., 2016;Zhang et al., 2021), but it was important to confirm this in intact Arabidopsis cells as localization for MTP8 homologs has been shown to vary.
We also confirm that overexpression of MTP8 in Arabidopsis confers enhanced tolerance to elevated Mn levels (higher biomass and chlorophyll per seedling) under basal Ca and also show this in low Ca conditions. MTP8-overexpressing plants have previously shown increased tolerance to moderate Mn toxicity (Chu et al., 2017) and enhanced Mn accumulation in root vacuoles (Eroglu et al., 2016).
Taken together, these findings support the hypothesis that the hypertolerance to Mn toxicity conferred by MTP8 overexpression is due to enhanced Mn sequestration within the vacuole, enabling greater resistance to cytoplasmic toxicity and thus enhancing growth ( Figure 10). This mechanism is also in agreement with results for Glycine max (soybean) MTP8, which also confers resistance to elevated Mn when expressed in Arabidopsis (Li et al., 2021). In this case, GmMTP8 was localized to the ER, although localization was not shown in plants that were conferring Mn tolerance (Li et al., 2021). In contrast, although tea CsMTP8 confers increased tolerance in Arabidopsis, it is proposed to function in Mn transport out of the cell as it is localized to the plasma membrane and reduces Mn accumulation (Li et al., 2017).
Nevertheless, whichever mechanism, the results suggest that developing crops with enhanced expression of MTP8 could be beneficial in enabling survival and improving yield when grown under poor soil conditions.
3.6 | Heterologous expression in yeast supports a Mn-transporting role for Arabidopsis Group 8/9 CDFs MTP10 and MTP11 both showed a punctate, Golgi-like pattern in yeast consistent with the localization seen in plants. Unlike the tonoplast localization in planta, we found that MTP8 localized to a punctate endomembrane compartment in yeast. A similar mislocalization has been observed for ShMTP8, which also targets the tonoplast in Arabidopsis and tobacco, but localizes in this case to the ER in yeast (Delhaize et al., 2003). It is possible, therefore, that the sorting signals for some of the Mn-MTPs may not be recognized appropriately in yeast, although the system is still extremely useful in testing for Mn transport properties. When compared directly, MTP8 and MTP10 conferred the greatest Mn tolerance to yeast mutant pmr1, whereas MTP11, although still providing tolerance, was not quite as effective.
MTP8 and MTP10 seem specific for Mn, whereas MTP11 also conferred slight Fe tolerance to ccc1, suggesting an ability to also transport Fe. In contrast, Chu et al. (2017) reported that MTP8, but not MTP11, was able to restore growth of ccc1. Here Fe was supplied at 5 mM, whereas Chu et al. (2017) supplied 3 mM Fe, so this may account for this difference.
Most hypothetical structural information for CDFs is based on the crystallized structure of Zn-transporting CDF, EcYiiP. EcYiiP functions as a homodimer with three Zn-binding sites, A, B, and C. Zn ions at site A are coordinated by the DxxxD and HxxxD motifs of TMDs 2 and 5, respectively, which form the DD-HD coordination site (Lu & Fu, 2007;Lu et al., 2009). These domains are conserved between all CDFs, suggesting this site is also conserved, although the exact residues vary between kingdoms . Based on an  (Chen et al., 2016), Os MTP11 (Farthing et al., 2017), and the human SLC30A10 (Nishito et al., 2016;Zogzas et al., 2016

| Growth of Arabidopsis plants
Arabidopsis thaliana plants were in the Columbia (Col) WT background. Soil-grown plants were grown as described in Menguer et al. (2013). To achieve soil of pH 7.2, soil was limed with 20 g/kg CaCO 3 and 12 g/kg NaHCO 3 ; pH was determined using 100 mM CaCl 2 .

|
Sequences of entry clones in pENTR/D-TOPO were confirmed and MTP8, MTP10, and MTP11 sequences were recombined with destination vectors (using Gateway LR Clonase II™ enzyme; Invitrogen).
All destination clones were confirmed with sequencing.

| Plate-based metal tolerance assay in Arabidopsis
Arabidopsis seeds were surface-sterilized in 15% (v/v) bleach for 20 min, rinsed five times with sterile water, and inoculated onto plates containing 0.8% (w/v) agarose (Melford Laboratories Ltd; www. melford.co.uk), 1% (w/v) sucrose (VWR Chemicals; uk.vwr.com), and either one-half-strength Murashige and Skoog medium (1/2 MS; Murashige & Skoog, 1962) or the same medium but with Ca levels ranging from 1.495 mM (standard Ca) to 0.1 mM CaCl 2 (low Ca) as described previously (Mills et al., 2008). For Mn assays, Mn was supplied as MnSO 4 at the indicated concentrations, and for Mn deficiency, no Mn salts were added to the media. Fe/pH experiments were set up as described in Eroglu et al. (2016). Seed were stratified at 4 C for 48 h prior to transfer to a controlled-environment cabinet (23 C, 16 h light; 18 C, 8 h dark; light intensity 100-120 μmoles m À2 s À1 ) with plates incubated vertically. Plants were grown for 21-24 days. Fresh weight (FW) and chlorophyll measurements were determined as described previously Mills et al., 2008), with generally six plates per condition, with four seedlings per genotype, per plate. Chlorophyll was determined following extraction in N,N-dimethylformamide (Moran, 1982; Sigma-Aldrich; www.sigmaaldrich.com). Experiments presented are representative of at least two independent experiments, and data are the means ± SE, on a per-seedling basis. All measurements were analyzed using one-way ANOVA or GLM at each concentration, with Tukey's post hoc test.

| Arabidopsis transformation
GV3101 Agrobacterium tumefaciens cells carrying plant expression vectors were used to transform Arabidopsis WT (Columbia), mtp8-2, and mtp11-1 plants using the floral dip method (Clough & Bent, 1998). Positive transformants were selected on ½ MS plates supplemented with 50 μg mL À1 hygromycin. Segregation analyses at the T2 and T3 stages were performed to isolate single-insertional homozygous transgenic plants. Metal tolerance assays and confocal microscopy studies were performed on homozygous T3 plants. To image, whole seedlings were placed on a microscope slide in water with a cover slip. Cell wall staining was performed with propidium iodide (Invitrogen), described in Menguer et al. (2013). Representative images are presented, after imaging several independent transgenic lines, with multiple seedlings imaged per line.

| Real-time PCR
Arabidopsis material was harvested for RNA extraction and cDNA synthesis as above. Quantitative real-time PCR (qPCR) was carried out in an Applied Biosystems StepOne real-time cycler using PrecisionPLUS Rox and SYBR Green Mastermix (Primer Design).
Relative expression levels of MTP8 and MTP10 were determined according to Pfaffl (2001) and normalized against UBQ10 as a constitutively expressed control. Primers are listed in Table S1. .5% (w/v) D-Glucose; 2 mM Na 3 PO 4 ; 60 mg/L acetosyringone in dimethyl sulfoxide). This Agrobacterium suspension was infiltrated into 4-to 6-week-old greenhousegrown tobacco plants (Nicotiana tabacum, Petit Havana), either WT or stably transformed with trans-Golgi-marker ST::RFP, as described in Brandizzi et al. (2002). ManI::GFP in the pBI221 vector (Shen et al., 2013) was used as a cis-Golgi co-expression marker. Infiltrated plants were grown normally for a further 48 h; 1-cm leaf-tissue discs were mounted in water on a microscope slide with cover slide and fluorescence observed using confocal microscopy as below. Representative images are presented, after at least three independent transfections per construct.

| Yeast transformation and metal-tolerance assays
Saccharomyces cerevisiae yeast strains used were: WT BY4741 and pmr1 (Euroscarf; www.euroscarf.de) for Mn-complementation analyses or zrc1 cot1 for Zn-and Co-complementation analyses; WT DY150 and ccc1 were used for Fe-complementation analyses. Full genotype information is listed in Menguer et al. (2013). Yeast transformation was performed using a LiOAc/PEG method (Gietz et al., 1992) selected on SC (synthetic complete) medium without uracil and with 2% (w/v) glucose, as described in Menguer et al. (2013).
Yeast cultures were inoculated overnight at 30 C in 5 mL SC glucose without uracil then to induce expression of genes of interest; cultures were resuspended in SC media without uracil, with 2% (w/v) galactose in place of glucose, and incubated for a further 4 h before dilution to OD 600 = 0.4. Further serial dilutions of 1/10 and 1/100 were also made. Seven μL culture was dropped onto 2% agar (w/v) plates containing SC galactose medium without uracil, supplemented with a range of metal concentrations (MnCl 2 , ZnSO 4 , CoCl 2 , FeSO 4 ; all Sigma-Aldrich). Plates were incubated at 30 C for 5 days.
To induce expression, cultures were grown as above. Cultures were fixed by resuspending in 100 μL 4% paraformaldehyde at room temperature for 15 min, before washing twice and resuspending in 100 μL of a 1 M KH 2 PO 4 /1 M K 2 HPO 4 /2 M sorbitol mix. Three microliter cells were positioned on a microscope slide with cover slip and imaged as below.

| Phylogenetic and sequence analysis
Multiple sequence alignments were performed by Clustal Omega (Sievers et al., 2011). Transmembrane domains were predicted using AramTmConsens, a consensus transmembrane alpha helix prediction program that combines output from 18 individual prediction programs, available on the ARAMEMNON database (Schwacke et al., 2003). For phylogenetic analysis, sequences for protein homologs to Arabidopsis MTP8-MTP11 were obtained from: Populus trichocarpa (poplar) and Sorghum bicolor (sorghum), Gustin et al. (2011); Beta vulgaris spp. maritima, Erbasol et al. (2013); O. sativa (rice), Chen et al. (2013); HvMTP8 and HvMTP8.1 from H. vulgare (barley), Pedas et al. (2014). Arabidopsis and rice sequences were used to search for homologs in B. rapa, Brachypodium distachyon, and Zea mays on Phytozome v9.1 (Goodstein et al., 2012), Cucumis sativus and Vitis vinifera on EnsemblPlants (Howe et al., 2020) and in barley on the International Barley Sequencing Consortium database (Schulte et al., 2009). The HvMTP11 coding sequence was predicted from the HvAK372762.1 contiguous sequence. A phylogenetic tree was reconstructed with Neighbor-Joining method, performed using MEGA (Molecular Evolutionary Genetics Analysis) 7 package (Kumar et al., 2016) with the following parameters: 1000 bootstrap replicates, pairwise deletion, and Poisson correction. Sequence data from this article can be found in data libraries under accession numbers listed in Table S2.
Hypothetical structural models for MTP8 and MTP11 were constructed using Swiss-Model (Biasini et al., 2014), with EcYiiP as homology template. Structures were visualized using The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC.

AUTHOR CONTRIBUTIONS
Lorraine E. Williams conceived the study. Emily C. Farthing, Kate C. Henbest, Tania Garcia-Becerra, and Kerry A. Peaston conducted the experiments. Lorraine E. Williams, Emily C. Farthing, and Kate C. Henbest analyzed the data and wrote the paper.